Investigations on A. laboriosa were conducted in the Himalayas of Nepal in November and December 1999. A. laboriosa nests were observed on two cliff sites. One rock cliff with 17 nests was located on the slope of Annapurna mountain in Kyumi, near Landrung at Modi Khola river in Kasaki district (long. 83°50′ E., lat. 28°22′ N., alt. 1,200 m.). The other rock cliff with 53 nests was situated in Chale at Koshi river near the village of Chaku in Sindhupalchok district (long. 85°48′ E., lat. 28°09′ N., alt. 1,500 m) near the Tibetan border. All the colonies were observed from the other side of the rivers from a distance of 28 m. In Chaku, three additional colonies were observed to which we had direct access and their activities were recorded from a distance of 0.25–1.5 m.

The defense behavior of all the colonies was observed during several days continuously from 0800 to 1600 hours while investigating periodic mass flights (Woyke et al. 2003).

A. d. dorsata colonies were observed in Nepal during 5 months from January to May 1999 and during 8 months from October 1999 to June 2000. Five sites with a total of 141 nests were observed at the Tribhuvan University campus in Rampur, Chitwan district, southern Nepal (long. 84°21′ E, lat. 27°39′ N., alt. 190 m) and the vicinity. Fourteen full day observations were made at the University on three sites with 7, 19 and 65 A. dorsata nests while investigating periodic mass flights at weekly intervals (Woyke et al. 2004a). The nests were observed from a distance of 4–12 m. We had also direct access to several colonies where the bees could be observed and recorded from a close distance.

Colonies of A. d. dorsata were also observed in Bangalore, India. The investigations were conducted for 11 days from 3 to 13 March 2002. Greater congregations of A. dorsata colonies were observed in two nest sites, one consisting of 26 nests at the polytechnic building in the city and the other of 82 nests on a banyan tree (Ficus bengalensis) at the campus of Agricultural University. The nests were observed from a distance of 1.5 and 8 to 10 m. All the colonies were observed continuously from 0800 to 1800 hours. We had close access to four nests.

Close observation on two nests of A. d. breviligula were conducted in The Philippines in Los Baños and in Alfonso from 27 February to 6 March 2004.

Bee colonies located at inaccessible places were examined with the aid of 12 × 50 binoculars. The activities of workers were recorded with a video camera with 24× optical zoom. Close observations and video recording were made from a distance of 0.25 to 1.5 m.

Defense behavior of individual worker bees was stimulated by free flying hornets, by killed hornet or dummy hornet fixed to a wire and also by different other objects. The disturbing objects were moved in front of the nests at a distance of 10 to 30 cm. In addition, the defense behavior was also observed while we were working directly with the colonies. We exchanged pieces of brood combs from A. d. dorsata and A. mellifera and A. cerana (Woyke et al. 2001), or we investigated hygienic behavior (Woyke et al. 2004b, c).

The ambient temperatures were measured every hour while conducting other studies (Woyke et al. 2004d, 2006). In The Philippines, in addition to ambient temperature also temperature of the outer layer of the nest curtain was measured with the aid of a thermocouple inserted in the outer layer of the curtain.

Defense body movements of individual worker bees was observed and analyzed from close up video recording. Close up video records of worker bees performing defensive body movements were made from lateral body side. The video was displayed at TV screen 43 × 56 cm where the length of the bee was 9 cm. The film was displayed at very slow mode (video frame after frame). The speed of successive frames was 1/25 s = 0.04 s. The TV display could be stopped at any frame. The workers moved their body up and down. Depending upon the distance and the angle of video recording, some bees visible on the TV screen were blurred. However, bees recorded perpendicularly to their bodies were clear, and we were able to select many clear and sharp images of bees. When the up movement reached its maximum, it stopped before changing into the down direction. We were interested in the maximal inclination when the body stopped to move. When the right video frame was found, the display was stopped. We selected clear and sharp images of body position in its maximal deflection. We were interested in two traits, maximal body inclination and the duration of particular body movement phases. The inclination was measured in relation to the vertical. At this frame stop, the drawings were also made. For that purpose, a transparent paper was fixed to the TV screen and the drawing was made.

The duration of particular phases of body movement was measured after the number of video frames was counted in which the phenomenon occurred and next it was multiplied by 0.04 s (frame succession speed 1/25 s = 0.04 s). Measurement of inclination or duration was repeated in each bee subspecies 30 or 45 times and all together 540 measurements were made. [Inclination: 30 repetitions × three species × three inclination measurements (thorax, abdomen, tip) = 270. Duration: 45 repetitions × two duration (up and down) × three species = 270. Together 270 + 270 = 540]. Altogether more than 1,000 video frames were individually checked.

ANOVA was applied and LSD was used to determine the significant differences between the means.

Video clips recording hornet hunting near A. laboriosa nest, defense body movements of individual workers of the three bee subspecies and defense waving on curtain nests of the colonies are presented in the online version. Due to transformation from PAL to *. avi, format and due to small size, many details described by us are not visible at the clips. We made all observations and all measurement on the original images recorded at the tape in Pal format and displayed at TV screen.

Undisturbed A. laboriosa and A. dorsata bees hang in the outside layer of the curtain covering the comb, head upwards. The anterior part of the body is inclined toward the comb by an angle of about 5° with respect to the vertical (Fig. 2a). The wings are spread apart. In A. laboriosa forewings were spread under an angle of about 55°, and the hind wings were spread more, under an angle of about 160° (Fig. 1a). In A. dorsata, forewings were spread more than in A. laboriosa under an angle of about 75°, however hind wings were spread less, under an angle of about 145° (Fig. 1a). Some A. dorsata workers in the curtain did not spread hind wings more than the forewings, but kept them spread under the forewings under an angle of only 80°. In this case, the forewings were spread under larger angle (about 95°) than in the other worker (75°) bees (Fig. 1b).

Fig. 1 Dorsal view of the position of wings during particular stages of defense body twists in two species of the giant honey bees A. dorsata and A. laboriosa. a Resting position, b alert position in A. laboriosa and resting position in A. dorsata, c position at maximal body twist.

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At lower temperature, the bees lift and lower the abdomen between spread wings, performing temperature dependent, dorso-ventral abdomen flipping (AF; Woyke et al. 2004d, 2006). No any thorax or wing movement occurs.

When a hornet was hovering near the nest, some of the near by bees took an alert position. They turned their heads and thoraces toward the hornet. The inclination was about 10° to 15° with respect to the vertical (Fig. 2b). Contrary to the resting bees, the tip of the abdomen of alerted bees was deflected dorsally. All alerted bees folded the wings over the abdomen (Fig. 1b, alert position). Any movement of the hornet triggered defense body movement of the nearby worker bees. The bees turned their bodies in the vertical direction. The turning axis passes through the thorax. The thorax rotated by an angle of about 60°. While the thorax rotated, the head was lowered and the abdomen and the wings were lifted up (Fig. 2c,d). Thus, really, only the thorax rotated and as a result of this, the head was lowered and the abdomen, due to high speed, was trust up to about 75° in relation to the vertical (Fig. 2d). The wings remained all the time over the abdomen, however they sprayed a little more (angle about 45°, Fig. 1c), than during the alert position (Fig. 1b). At the maximal inclination of the abdomen, the wings of all the bees performing DBM were positioned above the abdomen (Fig. 2d). However, the deflected abdominal tips, of part of the bees, protrude over the wings. No any stroke down of wings occurred during the DBM. Next, the whole body turned back to the resting position (Fig. 2f). If the disturbance continued the DBM were repeated as long as the threat operated.

Fig. 2 Lateral view of body position during successive stages of defense body twist in three subspecies of the giant honey bees. a Resting position, b alert position, c defense body twisting, d position at maximal body twist, e start of returning to resting position, f resting position, g, h some unusual position in A. dorsata. The legs are not proportional to the body.

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The term defense body movement does not specify the type of the movement. Therefore, we recommend using the term defense body twisting (DBT), which suggests (fort and beck) up and down turns of the body.

When a hornet approached A. dorsata nest, then an alert position was also noticed, however it differed from that of A. laboriosa. The body position was similar (Fig. 2b). However, contrary to A. laboriosa, none A. dorsata worker bee folded the wings over abdomen. Wings of most of the bees remained extended. Many bees kept hind wings extended more than forewings (Fig. 1a). However, some bees extended hind wings less then forewings (Fig. 1b). Any disturbing movement of the hornet triggered DBT. However, significant differences occurred. At the beginning, A. dorsata bees lifted the tip of the abdomen between the extended wings. The tip was deflected more or less upwards. The anal part of the abdomen was lifted higher than the wings (Fig. 2c and d). Such phenomenon newer occurs in any A. laboriosa bee. Those few bees, which had the wings folded over the abdomen, extend them now. Next, the bee rotated the thorax by about 65°. At the same time, the head was lowered and the abdomen, due to high speed, was trusted up to about the horizon position and the tip was deflected more (Fig. 2d). While the thorax rotated, the wings were lifted up and were extended apart more, to an angle of about 120° (Fig. 1c). Extension of the wings varied. Sometimes forewings extended as much as they covered the hind wings. Thus the wings of all A. dorsata bees were extended at the maximum DBT much more than in A. laboriosa (Fig. 1c). All movements of the abdomen of A. dorsata were performed between spread wings. Contrary to A. laboriosa, the wings of all A. dorsata bees were lifted lower, than the dorsal surface of the abdomen. As a result, at the maximal inclination of the abdomen, the wings of all A. laboriosa were positioned above the abdomen, and of all A. dorsata below the dorsal surface of the abdomen (Fig. 2e). Next the bee turned the body back to the initial position (Fig. 2f). DBTs were repeated as long, as the threat operated.

Variations were found in DBT of A. dorsata worker bees. Few workers lifted only the abdomen, but did not turn the thorax. Wings of such bees were not lifted, but were kept down in the initial position, during the whole DBT (Fig. 2g). Such behavior was never detected in any A. laboriosa bee. Some other A. dorsata bees turned the thorax already at the beginning of the DBT and lifted the wings and the abdomen already at the initiation of the DBT (Fig. 2h). All this shows that raising and lowering the wings is closely related with twisting the thorax.

All this above shows that several considerable differences were found in the DBT between both species. The differences did not concern some bees of both species, but all or none.

Close observations did not reveal any distinct differences in DBT of individual worker bees to stimuli from free flying hornet, killed hornet, dummy hornet or other moving objects.

Highly significant differences were detected in all traits between the tree subspecies. The total cycle of DBT in A. d. breviligula was the most vigorous, lasting only 0.11 s. The duration of DBT in A. d. dorsata (0.26 s) was twice as long and in A. laboriosa (0.33 s) three times as long. It was noticed that the duration of DBT was longer in those individual bees, which raised the abdomen higher.

December 6, 1999 in Chaku (Himalayas near Tibet), the day when DBT of A. laboriosa was video recorded, ambient temperature (t _a), varied, between 1100 and 1500 hours, from 15 to 17°C. During proper video recording, 1100–1130 hours, the t _a was 15°C.

December 20, 1999 in Rampur (Chitwan, Nepal), when DBT of A. d. dorsata was recorded, t _a varied, between the same hours, from 18 to 26°C. During recording 1200–1230 hours, the t _a was 20°C.

March 1, 2004 in Los Baños (The Philippines), when DBT of A. d. breviligula was recorded, t _a varied, between the same hours, from 35 to 37°C. Here also temperature of the outer layer of the curtain (t _c) was recorded. It varied between the same hours from 34.9 to 35.4°C. The difference between minimum and maximum t _a was here 2°C while between t _c only 0.5°C. The low difference in t _c is due to the ability of the bees to regulate curtain temperature and to keep it quite stable. During proper recording, 1100–1200 hours, t _a varied from 35 to 36°C and t _c from 34.9 to 35.3°C.

Despite the low difference between t _a in Chaku and in Rampur (5°C), the angel of rotating the thorax and raising the abdomen by A. laboriosa and A. d. dorsata differed highly significantly (Table 1). Contrary, despite the high difference between t _a in Rampur and Los Baños (16°C), rotating the thorax and raising the abdomen by A. d. dorsata and A. d. breviligula did not differ highly significantly. All the results indicate that the differences in DBT between the three bee subspecies were not related to differences in t _a between the three places. This suggests that the differences in DBT behavior are characteristic for those subspecies.

During the 2 days of continuous close observations in Chaku, (5 and 6 December) the defense behavior of A. laboriosa workers against a hornet hunting near the nest was video recorded (presented in the online version). The hornet was flying at a distance of 5–20 cm from the curtain surface. It was hovering head outside the nests watching for returning foragers. Most of returning foragers changed the direction of flight and avoided of being caught. The hornet was most successful in catching returning bees during periodic mass flight (Woyke et al. 2003). However, early in the morning, when the temperature was low (7°C to 9°C) and the bees were not flying, the hornets picked the workers directly from the lower edge of the nest curtain.

At higher temperature the neighboring workers surrounding the center of the disturbance followed the DBT with a delay of 0.04 s. This resulted in the circular defense waves (DW) spreading over the curtain of the nest. We observed the DW behavior of A. laboriosa colonies several thousands times. One quick pass of an intruder like an insect, bird or even a falling leaf near A. laboriosa nest triggered normally only one defensive wave. A longer pass generated two or three waves. However, hunting of a hornet near the nest differed essentially from other disturbances. The hornet was hovering near A. laboriosa nest for a dozen of minutes. As long as the hornet did not change its position, the bees did not make any DBT. However, any approach (backwards) of the hornet to the curtain, or change of the position in vertical or horizontal plane, triggered new DBT (presented online). As a result, we observed 40 DW per 1 min. Occasionally, when a second hornet approached the nest for hunting, the circular DW over the curtain expended from two different centers. As a result, the number of circular waves increased to 70 during 1 min. As soon as the hornet flew away, the workers stopped the defensive behavior. The hunting hornets disturbed the colonies almost continuously throughout the day.

We also provoked defense behavior many times by approaching a pencil, pocket knife, finger, hand, stick or photo- and video camera. Each approach of those objects triggered one DBT and one DW. Each movement resulted in a new DBT. The number of DBT did not depend upon the size of the disturbing object nor its nature, like live, dead or dummy hornet. As soon as the disturbing object was removed, no any more DWs were performed, although individual workers could make some DBT.

We conducted similar observations on A. dorsata nests in India. Our results were similar to various published already. However, we must add that in A. dorsata also as soon as the hornet left the colony, the bees of the curtain stopped to perform DW.